The Norrin/Frizzled4 signaling pathway in retinal vascular development and disease

Abstract

Disorders of retinal vascular growth and function are responsible for vision loss in a variety of diseases, including diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity and retinal artery or vein occlusion. Over the past decade, a new signaling pathway that controls retinal vascular development has emerged from the study of inherited disorders - in both humans and mice - that are characterized by retinal hypovascularization. This pathway utilizes a glial-derived extracellular ligand, Norrin, that acts on a transmembrane receptor, Frizzled4, a coreceptor, Lrp5, and an auxiliary membrane protein, Tspan12, on the surface of developing endothelial cells. The resulting signal controls a transcriptional program that regulates endothelial growth and maturation. It will be of great interest to determine whether modulating this pathway could represent a therapeutic approach to human retinal vascular disease.

Figure 1

Retinal oxygen consumption and the microanatomy of the retinal vasculature. (a) Relative oxygen consumption in the rat, per gram of tissue [1]. (b) Oxygen levels at different depths within the dark adapted cat retina [5]. (c) Left, the human eye in cross-section; right, the anatomy of the retinal and choroidal vasculatures (red). The retinal vasculature spans the GCL and INL; the choroidal vasculature is immediately beyond (to the right of) the RPE. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segments; NFL, nerve fiber layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segments; RPE, retinal pigment epithelium.

Figure 2

Development of the retinal vasculature. (a-c) Three successive stages in ocular development are shown from left to right. In humans, these stages occur in utero; in mice, they occur during the first two postnatal weeks. The retinal vasculature grows out from the optic nerve along the vitreal surface of the retina (a) before sending secondary branches into the retina to form the outer and then the inner layers of capillaries (b and c). Regression of the fetal vasculature within the vitreous and on the posterior surface of the lens (b and c) accompanies development of the retinal vasculature. (d) An endothelial tip cell displays numerous long filopodia; this morphology closely resembles that of an axonal growth cone. (e) In the early postnatal mouse retina, ECs (green) migrate along a network of astroctyes (red) as the developing vasculature advances across the vitreal surface of the retina. Abbreviations: NBL, neuroblast layer.

Figure 3

Ophthalmoscopic images of the human retina. (a) Fundus photograph of a normal retina showing the origin of the retinal vessels at the optic disc (left) and the dorsal and ventral sweep of the major arteries and veins above and below the fovea (right). (b) Fundus photograph of a retina from a FEVR patient showing a horizontal retinal fold (white arrows).

Figure 4

Norrin/Fz4 signaling in the retinal vasculature. (a) Proteins at the EC membrane that mediate Norrin/Fz4 signaling. The Lrp5 coreceptor (blue) has a large extracellular domain with four tandem copies of the YWTD-propeller and EGF domain (ovals), three tandem copies of the low density lipoprotein receptor domain (rectangles), a single transmembrane domain, and a cytosolic carboxy-terminal domain. Fz4 (red) has an amino-terminal ligand binding cysteine-rich domain (ball) followed by a GPCR-like integral membrane domain with seven transmembrane segments. Tpan12 (green) has four membrane spanning segments. Norrin (brown) is a disulfide-linked dimer. (b) Defects in retinal vascular development in the absence of Norrin/Fz4 signaling. Cross sections of adult mouse retinas of the indicated genotypes, with endothelial cells (red) visualized with GS lectin staining and nuclei (blue) visualized with DAPI staining. In the WT retina, the vasculature can be seen at the vitreal surface of the retina, and on either side of the INL. In the two mutant retinas, vertical white arrows show two clusters of ECs that have penetrated partway into the retina from the vitreal surface. (c) Flatmounts of adult WT or Fz4 KO mouse retinas with the blood vessels visualized by intravascular filling with fluorescein-dextran. The major arteries and veins can be seen emerging from the optic disc in the upper left of each image. Vertical white arrows show two of the many clusters of ECs that have penetrated partway into the retina from the vitreal surface. Abbreviations: CC, choricapillaris, i.e. the choroidal vasculature.